DIFFERENTIAL EXPRESSION OF DIVERSE PLANT ACTIN GENES

多种植物肌动蛋白基因的差异表达

• 批准号: 2391996
• 负责人: Richard Brian Meagher
• 金额: $ 23.62万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391996
• 关键词: Arabidopsis; actin binding protein; actins; antisense nucleic acid; fusion gene; gene induction /repression; gene mutation; genetic mapping; genetic promoter element; intermolecular interaction; introns; messenger RNA; plant genetics; polymerase chain reaction; protein structure function; reporter genes; site directed mutagenesis; structural genes; tissue mosaicism; transposon /insertion element; yeasts

Our research is focused on the hypothesis that the ancient and divergent classes of plant actin genes have been preserved throughout vascular plant evolution because they have unique patterns of gene regulation and/or encode proteins with unique functions. During the last grant period we isolated, sequenced, and characterized the ten members of the Arabidopsis actin gene family (ACT1, 2, 3, 4, 5, 7, 8, 9psi, 11, & 12). They fall into six subclasses which were approximately as divergent as vertebrate muscle actin is from cytoplasmic actin, but the plant proteins had much higher levels of charged residue variability. Based on gene specific Northerns and RT-PCR analysis of RNA expression and 5' region/reporter fusions examined in hundreds of independent transgenic plants, eight of the genes in five subclasses were expressed at very high levels in at least one cell type. These five subclasses had very distinct and different expression patterns covering all the tissues and developmental stages of Arabidopsis (e.g. the ACT2/ACT8 subclass was constitutive in vegetative tissues, while the ACT1/ACT3 subclass was expressed in organ primordia and mature pollen). An initial characterization of Arabidopsis profilin sequences demonstrated the presence of a large and complex gene family which included constitutive (PRF1, 2, 3) and pollen specific members (PRF4). In light of these and other data on the diverse plant actins and profilins we propose a second hypothesis for our future work: there are concordantly expressed subclasses of actins and actin binding proteins (e.g. profilins), which have evolved distinct protein-protein interactions. Based on these two hypotheses our specific aims for the next grant period are: (l) to demonstrate that the various actin genes or subfamilies are required for normal Arabidopsis growth and development by characterizing the phenotype of plants deficient in actin expression (from a T-DNA insertion library and by antisense suppression and co-suppression); (2) To determine if the major cis-elements controlling tissue specific expression are in the promoter region, mRNA leader or leader intron; (3) to determine the detailed expression patterns of a few representative members of the diverse profilin subclasses (i.e. PRF1 and PRF4); (4) to initiate experiments which will connect the origin of the diverse actin lineages with macroevolutionary events (e.g. ACT1 with the origin of meristems); and (5) to compare the strength of protein-protein interaction between actin-profilin pairs that are co-expressed, with those that are not. This work will expand our basic understanding of the eukaryotic cytoskeleton and enhance our ability to manipulate the properties of higher plants.

我们的研究重点是古代和不同的假设 植物肌动蛋白基因的类别已保存在整个血管植物中 进化是因为它们具有基因调节和/或的独特模式 用独特功能编码蛋白质。在最后一个赠款期间我们 隔离，测序和表征拟南芥的十名成员 肌动蛋白基因家族（ACT1、2、3、4、5、7、8、9PSI，11和12）。他们掉进了 六个子类大约与脊椎动物肌肉不同 肌动蛋白来自细胞质肌动蛋白，但植物蛋白的蛋白质高得多 电荷残留变异性水平。基于基因特定的北方 RT-PCR的RNA表达和5'区域/报告基因融合 在数百种独立的转基因植物中检查了八个基因 在五个子类中，在至少一个单元中以非常高的水平表达 类型。这五个子类具有非常不同和不同的表达 涵盖拟南芥的所有组织和发育阶段的模式 （例如，ACT2/ACT8亚类是在营养组织中的组成型 ACT1/ACT3子类在器官原始和成熟 花粉）。拟南芥酸蛋白序列的初始表征 证明存在一个大而复杂的基因家族 包括本构（PRF1、2、3）和花粉特定成员（PRF4）。在 这些有关植物肌动蛋白和profilins的数据和其他数据，我们 为我们的未来工作提出第二个假设：有一致的 表达肌动蛋白和肌动蛋白结合蛋白的亚类（例如 profilins），它们已经进化了不同的蛋白质 - 蛋白质相互作用。 基于这两个假设，我们对下一个赠款期的具体目标 是：（l）证明各种肌动蛋白基因或亚家族是 正常拟南芥生长和发展所需的 植物缺乏肌动蛋白表达的表型（来自T-DNA 插入文库以及反义抑制和共抑制）； （2）至 确定控制组织特异性表达的主要顺式元素是否 在促进者地区，mRNA领导者或领导者内含子； （3）确定 少数代表成员的详细表达模式 多样的profilin子类（即PRF1和PRF4）； （4）启动 将连接各种肌动蛋白谱系的起源的实验 随着宏观进化事件（例如ACT1具有分生组织的起源）； （5）比较蛋白质 - 蛋白质相互作用的强度 肌动蛋白 - 易生蛋白对共表达，而不是那些。这 工作将扩大我们对真核细胞骨架的基本理解 并增强了我们操纵高等植物特性的能力。